Adenovirus E1A studies have contributed significantly towards understanding viral gene regulation and tumorigenesis. The modular nature of the E1A coding region has made it possible to examine these functions independently. The CR3 zinc finger region of E1A serves to activate transcription by interacting with three cellular proteins: TBP, hTAF135 and the newly discovered mediator protein, hSur-2. Mutational analyses and binding studies suggest that these three proteins require different residues of CR3 for activation. Solution of the 3-D structure of the CR3 region alone and complexed to these individual target proteins will provide the first detailed view of the E1A activation complex. In Ad12-tumor cells, a different region of E1A mediates down-regulation of MHC class I transcription enabling these tumor cells to escape lysis by cytotoxic T lymphocytes. This shut-off of MHC class I transcription is regulated by two factors (NF-KB and COUP-TFII) that affect the class I enhancer. NF-KB (p65/p50) fails to bind the class I enhancer and activate transcription because the p50 subunit is hypophosphorylated. This finding is novel in that it is the first example to show that p50 can regulate DNA binding of NF-KB and control its ability to activate transcription. To unravel this mechanism, the phospho-residues of p50 that regulate DNA binding will need to be defined and the cellular kinase identified. By contrast, COUP-TFII binds strongly to the class I enhancer and represses transcription through its association with a HDAC complex that presumably causes chromatin compaction. This is the only known example in which a COUP-TF protein has been shown to regulate the MHC class I promoter. It will be essential to define the components of the COUP-TFII DNA complex by chromatin histone immunoprecipitation assays and to investigate whether Ad12 E1A is one of these components. In addition, a new understanding of Ad12 E1A mediated tumorigenesis will be acquired from analyzing genes which have been shown to be differentially expressed in Ad12 tumorigenic vs Ad5 non-tumorigenic cells. A recent discovery revealed that in addition to mediating MHC class I down-regulation, Ad12 EIA encodes a second tumorigenic function that is contained within a 20 amino acid region called the Spacer. Mutation of the Spacer prevents tumorigenesis without interfering with class I down-regulation. Screening a phage display library against the Spacer identified a protein with homology to a Natural Killer (NK) cell protein NKTR-l, consistent with the ability of Adl2-tumor cells to resist NK lysis. To investigate Spacer function, wt and mutant E1As will be tested for binding to NKTR-1. Finally, Wt E1A and mutant Spacer E1A cell lines will be compared for resistance to NK lysis and for differential gene expression by microarray analysis.